I have performed RT-PCR for exosomal miRNA. For normalisation I've used U6. But Ct value of the target gene is less than the normalised gene (U6). U6 is giving a Ct value of approx 32 whereas target gene Ct value is approx 29 . Should the Ct value of U6 be less than the target gene? Please share your views.
If your CT for your target is lower than your reference, all this means is that you have a higher copy number of your target than your reference. There is no need to think that U6 will always be the most abundant miRNA in a sample.
If your CT for your target is lower than your reference, all this means is that you have a higher copy number of your target than your reference. There is no need to think that U6 will always be the most abundant miRNA in a sample.
Jonathan's right, assuming that both primers have similar efficiency. The relative expression of the reference and target don't matter as much as the relative efficiency of the two primer sets, especially if you're quantitating your results using the (delta)(delta)Ct method.
I agree with the answers given above. As ling as negatives are negative and yr U6 is present in all the samples you should be good to go..
In principle, I agree with the others. But you must always bear in mind that Ct values above 30 might be unreliable due to the fact that below a certain level of mRNA/miRNA in your sample you won't get a normal distribution anymore but a Poisson distribution (see Rutledge and Stewart, PLOSOne, 2010, Figure 9). As long as your technical replicates are more or less constant, it should be o.k. even if your reference gene has low Ct values.
Greetings, Britta
You may wanna test which ref gene is best for you. I dont think U6 is working for you because of the high Ct value. Shoot for ref gene with CT value between 15 and 20.
Hope you got it working. Johannes
- Badges
- Science topic